NANO LETTERS

Artificial Organelle: ATP Synthesis from Cellular Mimetic Polymersomes

Hyo-Jick Choi and Carlo D. Montemagno*
Department of Bioengineering, Room 7523 Boelter Hall, 420 Westwood Plaza, UniVersity of California, Los Angeles, PO Box 951600, Los Angeles, California 90095-1600
Received September 22, 2005; Revised Manuscript Received November 9, 2005

2005 Vol. 5, No. 12 2538-2542

ABSTRACT
A complex cellular process was reconstructed using a multiprotein polymersome system. ATP has been produced by coupled reactions between bacteriorhodopsin, a light-driven transmembrane proton pump, and F0F1-ATP synthase motor protein, reconstituted in polymersomes. This indicates that ATP synthase maintained its ATP synthesis and therefore its motor activity in the artificial membranes. This hybrid proteopolymersome will have wide application in a number of fields ranging from the in vitro investigation of cellular metabolism to the synthesis of functional smart materials.

Cells are the major functional units of all living organisms and contain the optimal machinery for performing diverse biochemical functions. The energy for many of their essential functions is provided by ATP synthesized in the cytosol, mitochondria, or chloroplasts. It powers most of the energyconsuming activities of the cell and regulates many biological pathways: metabolic reactions, bioluminescence, muscle contraction, and active transport. Because of this importance, much research has been focused on ATP since its discovery in 1929 to understanding of the ATP synthesis mechanism.1-4 The integrative fusion technology, nanobiotechnology, applies the principles of living systems and engineering to develop devices to gain higher-order functionality by mimicking subcellular level natural processes. To mimic the functions of cells, some fundamental biological structures, such as lipid membrane, membrane proteins, and membranebound organelles have been employed. The most successful work has been an ATP-powered motor with nanofabricated propellers linked to the genetically engineered protein F1ATPase.5,6 The rotational motion of F1-ATPase is coupled with hydrolysis of ATP. F1-ATPase therefore possesses energy transduction properties, being able to convert chemical energy to mechanical energy and vice versa. Because the force of its rotational motion is compatible, F1-ATPase has been suggested to be an attractive choice for powering nanoelectromechanical system (NEMS) devices to function in biological systems.7 However, the usefulness of this engineered ATPase motor is limited by the external supply of ATP. Therefore, the aforementioned importance of ATP in cellular biochemical reactions lends itself into a central role in future protein-based nanodevice fabrication. Hence,
* Corresponding author: e-mail, cdm@seas.ucla.edu; Fax, 310-794-5956. 10.1021/nl051896e CCC: $30.25 Published on Web 11/18/2005 2005 American Chemical Society

the realization of ATP production from artificial organelles may be used for powering ATP-driven nanoscale hybrid devices such as molecular motors and as a power source for performing biochemical reactions in vitro. The plasma membrane is a flexible barrier that is formed by the self-assembly of lipid molecules and serves to separate the cells internal environment from its external environment. Conventionally, lipid membranes and vesicles (liposomes) have been used for the recreation of the natural environment of membrane-bound proteins, as they provide the most logical and natural environment for observing membrane protein functionality. While they do have excellent assay characteristics, the future applicability of protein-reconstituted lipid vesicles (proteoliposomes) is intrinsically limited due to both chemical and mechanical weaknesses. This suggests to us that replacing traditional phospholipids with stable biomimetic membranes is key technology for the development of stronger hybrid devices. To create the artificial environment necessary for membranebound proteins, self-assembled, amphiphilic ABA triblock copolymers have been suggested as a potential building material for new biomimetic membranes.8-10 These polymers, composed of a hydrophobic layer sandwiched between two hydrophilic layers, are analogous to a typical lipid bilayer. Such artificial biocompatible polymer membranes have demonstrated the stability needed for long-term organic/ inorganic hybrid systems. Until recently, most of the previous work has focused on the measurement of a single pore proteins functionality after reconstitution. In a previous publication, we reported that the energy-transducing protein, bacteriorhodopsin (BR), can be incorporated into polymersomes and retain its photoactivity.11 These preliminary studies

inspired us to research a novel biotic/abiotic hybrid system involving multiple complex proteins. ATP synthase is a rotary motor protein which is composed of two domains, the membrane-integrated F0 and the soluble F1. F0 functions to conduct protons across the membrane by a rotational mechanism of the intramembrane subunits (rotor), resulting in a torque which is transmitted to the catalytic sites through the rotor stalk. F1 is responsible for the catalytic activity driving the synthesis and hydrolysis of ATP using mechanical energy. Coupling activity between the F0 and F1 complexes drives proton movement toward the F1 side of the membrane, resulting in ATP synthesis. Therefore, successful ATP synthesis depends on the proper mechanical and chemical environments enabling the rotor and stator to function like a rotary engine. In this Letter, we report the generation of ATP, by photoinduced phosphorylation from the coupled activity of BR and F0F1-ATP synthase reconstituted biomimetic polymersomes. ATP synthase maintained its rotating activity in the synthetic polymersomes and produced ATP utilizing the photoinduced proton gradient generated from BRs activity. This represents not only the first successful biosynthesis through coupled reactions between reconstituted transmembrane proteins in a single proteopolymersomes but also the demonstration of molecular motor functionality in a polymer membrane. It is expected that this ATP-producing proteopolymersome will enable not only propelling biomotors but also the development of ATP-driven nanoscale devices. Polymersomes were prepared with an amphiphilic triblock copolymer, PEtOz-PDMS-PEtOz [poly(2-ethyl-2-oxazoline)-b-poly(dimethylsiloxane)-b-poly(2-ethyl-2-oxazoline)].10 PEtOz-PDMS-PEtOz was synthesized by ringopening cationic polymerization of ethyl oxazoline with bifunctional benzyl chloride-terminated PDMS as previously described. This polymer vesicle has a wall thickness of about 4 nm, which is similar to a typical lipid bilayer thickness, and further supports its potential use as a replacement of traditional phospholipid bilayers. To achieve light-driven ATP biosynthesis, we reconstituted both BR and ATP synthase simultaneously into the polymersomes. BR is a light-driven proton pump that creates a proton gradient across the cell membrane via a light-induced BR photocycle.12-14 When coupled with ATP synthase, this proton gradient is used to synthesize ATP from ADP and inorganic phosphate (Figure 1).15-18 Our first experiments monitored BRs pumping activity in BR-proteopolymersomes. To observe the membrane morphology after protein incorporation, we performed transmission electron microscopy (TEM) analysis. As shown in the bright-field TEM image, small-sized polymersomes were observed distributed throughout the sample (Figure 2). Also, the polymersomes were mostly spherical in shape and no multilamellar vesicles were observed. From direct measurement of vesicle sizes by TEM micrographs, there is a narrow monomodal size distribution of vesicles with a mean vesicle diameter of 37 ( 12.2 nm. The generation of a photoinduced electrochemical proton gradient from both BR proteopolymersomes and BR-ATP
Nano Lett., Vol. 5, No. 12, 2005

Figure 1. Schematic representation of proteopolymersomes reconstituted with both BR and F0F1-ATP synthase. ATP synthase uses an electrochemical proton gradient generated by BR to synthesize ATP from ADP and inorganic phosphate (Pi).

Figure 2. TEM micrograph of BR-proteopolymersomes. A liquidnitrogen-cooled specimen stage was used to minimize the structural changes during observation.

synthase proteopolymersomes were measured by trapping 8-hydroxyprene-1,3,6-trisulfonic acid (pyranine), a fluorescent pH probe, inside the proteopolymersomes.18,19 Since the relative fluorescence intensity ratio at 402 and 456 nm (I456/ I402) is dependent on the hydrogen ion concentration, internal pH in proteopolymersomes can be measured by reading the characteristic peak intensities at 456 and 402 nm in the excitation spectrum. For measurement of light-driven internal pH changes, the fluorescence was first measured after incubation in the dark, and photoinduced intensity change was measured at different time intervals during continuous irradiation with a light source ( ) 570 nm). The conversion from I456/I402 to pH was performed as described in Hazard and Montemagno.18 As controls, we have shown the pH change inside polymersomes over time both in the presence and in the absence of light as well as the internal pH change of the proteopolymersomes without illumination.
2539

Figure 3. The proton pumping activity of BR-proteopolymersomes monitored by trapping 8-hydroxyprene-1,3,6-trisulfonic acid (pyranine) as a fluorescent pH probe inside proteopolymersomes. Light-driven pH change formed by BR-proteopolymersomes is shown together with those from control samples as a function of illumination time: illuminated proteopolymersomes (9), darkincubated proteopolymersomes (0), illuminated polymersomes containing no proteins (b), and dark-incubated polymersomes (O).

Figure 4. Photoinduced ATP synthesis in BR-ATP synthaseproteopolymersomes. ATP production per milligram F0F1-ATP synthase is shown as a function of illumination time: illuminated BR-ATP synthase-proteopolymersomes (9), dark-incubated BRATP synthase-proteopolymersomes (0).

The proton gradient formed by BR proton pumping activity was characterized in dark and light incubations (Figure 3). In the absence of light, no significant intravesicular pH (pH ) pHt - pH0, where pHt and pH0 are the measured internal pH values after t and 0 min, respectively) was observed from both proteopolymersomes and polymersomes without proteins. This indicates that the initial pH difference across the membrane is negligible. It is of note that polymersomes produced a small pH (0.03 units at 60 min) over the course of illumination. Considering that no protein is incorporated and polymersomes did not show any significant pH in the dark, we believe pyranine may undergo chemical modification during light incubation resulting in the observed pH. On the other hand, as can be seen in Figure 3 lightincubated proteopolymersomes generated a rapid decrease in intravesicular pH. After 60 min of light illumination, the internal pH change was measured to be about 0.09 units (in our experiments, the maximum pH measured was 0.12). This difference demonstrates that light-induced proton pumping by BR resulted in an internal pH decrease (pHt < pH0). We believe that the final pH value depends heavily on the statistical distribution of BR (i.e., orientation) in the membrane. The acidification of the proteopolymersomes indicates that more than 50% of BR in proteopolymersomes has a preferential orientation, i.e., with the C-terminus outside, resulting in an inward pumping of protons.20 That is, when more BR molecules are preferentially positioned in the proteopolymersomes to pump in the same direction, it is likely that a higher proton gradient can be achieved Also, the maximum pH was the same in the presence and absence of F0F1-ATP synthase (data not shown). Therefore, there was no evidence of increased proton permeability with the presence of uncoupled F0 in the membrane.21 A rapid acidification during initial illumination was followed by a relatively slow steady decrease in pH as shown
2540

in Figure 3. The results show that a maximum proton pumping activity of 10.4 10-3 pH min-1 occurred in the first 5 min of illumination. But, the rate rapidly decreased to about to about 4.8% (5.0 10-4 pH min-1) of the initial pumping rate between 20 and 40 min. After 40 min, further illumination resulted in a negligible change in pH (2.7% of the initial pumping rate, 2.7 10-4 pH min-1). The decreased proton pumping rate was presumably caused by both the back pressure effect22 and proton leakage through the polymer membrane (see Supporting Information), limiting the maximum attainable pH change. These fluorescence pH measurements demonstrate that BR molecules retained their native photoactivity in the polymersomes. Another important characteristic observed from proteopolymersomes is a longterm stability which is an important factor for developing biotic/abiotic hybrid devices. When stored at 4 C, the proteopolymersomes retained their proton pumping functionality for more than 3 months, which exceeded the 3-4 week lifespan of proteoliposomes. A quantitative determination of ATP concentration was performed with a bioluminescence assay using luciferin and luciferase.18,23,24 Luminescence occurs when luciferase catalyzes the oxidation of luciferin by consuming ATP. As the luminescence intensity is proportional to the amount of ATP present in the sample, we first prepared seven standard samples of known ATP concentration. On the basis of the measured luminescence intensities, we generated an ATP standard curve, with luminescence intensity versus ATP amount. The rotary catalytic ATP synthesis was demonstrated by reconstitution of ATP synthase together with BR. Figure 4 shows the ATP synthesis by BR-ATP synthase-proteopolymersomes after different lengths of light incubation. Dark-incubated proteopolymersomes did not show any synthesis activity. However, the result shows that ATP production markedly increased with the time of light incubation and reached 1140 nmol of ATP/mg of ATP synthase at 60 min. An interesting observation is that the BR-ATP synthase-proteopolymersomes did not exhibit the previously
Nano Lett., Vol. 5, No. 12, 2005

reported acceleration of ATP synthesis due to ATP binding to the enzyme.15 The maximum ATP synthesis rate was 116 nmol/min/mg of ATP synthase at the first 5 min. As ATP synthesis by ATP synthase is activated by the pH gradient across the membrane, it seems that the rapid acidification during the initial illumination has led to a higher initial ATP synthesis rate. Then, ATP production rate saturated to about 4.2 nmol/min/mg of ATP synthase for proteopolymersomes. In addition, as BR pumps protons inward, we concluded that F0F1-ATP synthase has a functional orientation with F1 facing outward. This is confirmed by the necessity of ADP and Pi to come in contact with the F1 subunit to produce ATP. The ability of the BR-ATP synthase-proteopolymersomes to synthesize ATP suggests that the triblock-copolymerbased cell mimics provide a stable biocompatible support for membrane proteins and other biological components. As proven by the preservation of ATP synthesis, complex functional requirements such as the rotating motion of the F0 subunit of ATP synthase as well as proton translocation were met by biomimetic polymer membranes. For these reasons, synthetic polymersomes are expected to perform a variety of functions to overcome various obstacles in the area of biomimetics. The present paper has shown that synthesized polymersomes can be used as artificial organelles. Although we have not assessed the optimal protein ratio, using these cellular mimics, we have recreated a biological ATP generation process in an artificial system and have demonstrated the feasibility of performing biosynthesis in polymersomes. Furthermore, light-driven protein functionality may depend on the relative composition of protein to amphiphile, which leaves room for further research.16 This analysis suggests that control over both specific orientation and optimal composition in proteopolymersomes is expected to prove the potential of proteopolymersomes as cell mimics that serve as a stable support where efficient biochemical synthesis can be replicated. Methods. BR was incorporated in the form of purple membrane (PM) which was obtained from Halobacterium salinarium grown for mass production.25 All samples were prepared and kept in the dark to preserve maximum proton pumping activity during assays. F0F1-ATP synthase was purified from Bacillus PS3 cells and concentrated as described in ref 18. PEtOz-PDMS-PEtOz triblock copolymer (Mn ) 7800, polydispersity index ) 1.48) was synthesized using the method described previously.10 To form polymer structures, the amphiphile was first dissolved in ethanol (0.1886 g/mL) and stirred at room temperature for 2 days to obtain a homogeneous solution. The 88.6 L polymer stock was then placed in an amber vial and 68.5 L of PM (BR concentration: 4.8 mg/mL) was added slowly dropwise to the vial. Stirring was continued for 1.5 h in the dark to ensure a homogeneous solution. Then, 27.7 L of ATP synthase (2.6 mg/mL) was added to the polymer/PM mixture, and the mixture was stirred for 30 min. A slow dropwise addition of this protein-polymer mixture into buffer solution (20 mM MOPS, Sigma; 50 mM Na2SO4, 50 mM K2SO4, 2.5 mM MgSO4, 0.25 mM DTT, Fluka; 0.2 mM
Nano Lett., Vol. 5, No. 12, 2005

EDTA, Sigma, pH ) 7.25) at the rate of 10 L every 30 s created BR-ATP synthase-proteopolymersomes. For pH measurement, 5 M pyranine (Molecular Probes) was added to the buffer solution when making polymersomes. Sample volume was controlled at 2 mL. The samples were then passed through 200 nm filter membranes to retain small unilamellar vesicles. The excess pyranine not encapsulated inside the polymersomes was removed via 48-h dialysis at 4 C in the dark. The formation of vesicles was confirmed by transmission electron microscopy (TEM) analysis. For TEM observation, the vesicle solution was dropped onto an amorphous carboncoated Cu grid by pipet. For faster drying, copper grids were placed on Kimwipes, and excess solution was removed by blotting. The samples were transferred to the TEM chamber using a liquid-nitrogen-cooled specimen stage to reduce structural changes of the specimen due to electron beam exposure. The specimen temperature was monitored to be -170 C during observations. The proton pumping activity of BR in the polymersomes was monitored using 8-hydroxyprene-1,3,6-trisulfonic acid (pyranine) as a fluorescent probe. Intravesicular pH was measured by reading fluorescence at the wavelengths of 402 and 456 nm. A 150 L portion of protein-polymer solution taken from all the samples was illuminated by 5.04 W green LED ( ) 570 nm) before measurement. An excitation scan with a luminescence spectrometer (LS 50B Perkin-Elmer) was performed from 350 to 475 nm at an emission wavelength of 511 nm. A xenon discharge lamp (20 kW for 8 s duration, pulse width at half-height <10 s) was used as an excitation source. Spectra were collected in 0.5 nm increments with an integration time of 0.5 s per point. Any external pyranine not removed by dialysis was quenched by the addition of p-xylenebispyridinium bromide (DPX, Molecular Probes). Small shifts in the excitation spectrum were corrected, and the conversion from fluorescence to pH was performed following the method described by Hazard and Montemagno.18 To measure ATP synthesis activity, a bioluminescence assay kit (FLAA Luciferin-Luciferase; Sigma) was used. A 50 L portion of Mg-ADP (0.2 M) and 25 L of Pi (1 M) were added to 500 L of proteopolymersome stock solution. Samples were continuously illuminated by a 5.04 W green LED ( ) 570 nm) to generate a proton gradient which is used by ATP synthase to produce ATP by phosphorylation of ADP. At various time points, aliquots of the reaction mixture were removed and vortexed with an equal volume of 8% trichloroacetic acid and analyzed for their ATP amount. ATP production was quantified by measuring the amount of light produced by the samples and fitting the results to a standard intensity vs ATP amount calibration curve. All experiments were performed at room temperature in dark. Acknowledgment. The authors thank Jeffrey Germain for helpful comments on various drafts of this paper and Hyeseung Lee for samples of purple membrane. We also wish to thank Cassanna Ouellette for the graphical representation of the proteopolymersomes.
2541

Note Added after ASAP Publication. There were errors in the units for the ATP synthesis rate and ATP production rate cited in the top left-hand column of the fourth page and an error in the reference 3 citation in the version published ASAP November 18, 2005; the corrected version was published ASAP November 23, 2005. Supporting Information Available: Description of the proton leakage through polymersomes. This material is available free of charge via the Internet at http://pubs.acs.org. References
Mitchell, P. Nature 1961, 191, 144. Cross, R. L. Annu. ReV. Biochem. 1981, 50, 681. Wang, J. H. Annu. ReV. Biophys. Bioeng. 1983, 12, 21. Boyer, P. D. Annu. ReV. Biochem. 1997, 66, 717. Soong, R. K.; Bachand, G. D.; Neves, H. P.; Olkhovets, A. G.; Craighead, H. G.; Montemagno, C. D. Science 2000, 290, 1555. (6) Bachand, G. D.; Soong, R. K.; Neves, H. P.; Olkhovets, A.; Craighead, H. G.; Montemagno, C. D. Nano Lett. 2001, 1, 42. (7) Montemagno, C. D. J. Nanopart. Res. 2001, 3, 1. (8) Meier, W.; Nardin, C.; Winterhalter, M. Angew. Chem., Int. Ed. 2000, 39, 4599. (1) (2) (3) (4) (5)

(9) Nardin, C.; Thoeni, S.; Widmer, J.; Winterhalter, M.; Meier, W. Chem. Commun. 2000, 15, 1433. (10) Choi, H.-J.; Brooks, E.; Montemagno, C. D. Nanotechnology 2005, 16, S143. (11) Choi, H.-J.; Lee, H.; Montemagno, C. D. Nanotechnology 2005, 16, 1589. (12) Oesterhelt, D.; Stoeckenius, W. Nat. New Biol. 1971, 233, 149. (13) Stoeckenius, W.; Lozier, R. H.; Bogomolni, R. A. Biochim. Biophys. Acta 1979, 505, 215. (14) Subramaniam, S.; Henderson, R. Nature 2000, 406, 653. (15) Richard, P.; Pitard, B.; Rigaud, J.-L. J. Biol. Chem. 1995, 270, 21571. (16) Pitard, B.; Richard, P.; Dunach, M.; Rigaud, J. -L, Eur. J. Biochem. 1996, 235, 779. (17) Groth, G.; Walker, J. E. Biochem. J. 1996, 318, 351. (18) Hazard, A.; Montemagno, C. Arch. Biochem. Biophys. 2002, 407, 117. (19) Kano, K.; Fendler, J. H. Biochim. Biophys. Acta 1978, 509, 289. (20) Seigneuret, M.; Rigaud, J.-L. FEBS Lett. 1988, 228, 79. (21) Okamoto, H.; Sone, N.; Hirata, H.; Yoshida, M.; Kagawa, Y. J. Biol. Chem. 1977, 252, 6125. (22) Hellingwerf, K. J.; Arents, J. C.; Scholte, B. J.; Westerhoff, H. V. Biochim. Biophys. Acta 1979, 547, 561. (23) Strehler, B. L. Methods Biochem. Anal. 1968, 16, 99. (24) Leach, F. R. J. Appl. Biochem. 1981, 3, 473. (25) Dencher, N. A.; Heyn, M. P. Methods Enzymol. 1982, 88, 5.

NL051896E

2542

Nano Lett., Vol. 5, No. 12, 2005